T-cell growth factor (TCGF), also termed interleukin-2 (IL-2), has been previously described and has been recognized as an important member of the family of lymphokines which are lymphocyte-derived regulatory proteins that play a role in cellular and humoral immune responses. TCGF is released from antigen-, phorbolester-or lectin-stimulated cultured T-cells and causes proliferation of primed T-lymphocytes and natural killer (NK) cells. lt facilitates the indefinite in vitro growth of functional T-cells and NK cells, and if injected into tumor-bearing animals, it may facilitate an elevated immune response to the tumor.
Because TCGF has been recognized for a number of years as an important protein with potential clinical use, there has been a substantial amount of research directed to characterizing its properties. Research in this respect has been hindered somewhat by the lack of a suitable procedure for obtaining meaningful quantities of truly pure TCGF. As a result, characterization of TCGF have often been at variance, undoubtedly due to the presence of contaminating proteins, the relative proportions of which depend upon the method of partial purification. Heretofore, there has been no generally useful method described which isolates a truly pure fraction of human TCGF in a quantity which permits its characterization, although analytical techniques, such as electrophoresis or isoelectric focusing, have been used to analyze impure protein fractions and may produce bands which contain minute amounts of pure TCGF.
Applicants herein have been studying TCGF since 1976, and for seven years had been somewhat frustrated in their research in their inability to obtain substantially pure human TCGF. Applicants attempted purifications based upon a variety of published techniques and also modified such published techniques in attempts to enhance the purity. In particular, applicants attempted to perform high performance liquid chromatography (HPLC) using available hydrophobic stationary phases, but without complete success. The highly hydrophobic stationary phases proved to have too great an affinity for TCGF, resulting in broadening of the elution peaks and inevitable overlap with peaks with those of contaminating proteins. Furthermore the high affinity of hydrophobic stationary phases for TCGF substantially reduced the percent recovery, that is, the total activity of TCGF in the eluted fractions as compared to the total activity of the TCGF in the material supplied to the HPLC column. Thus, there has been an existing need of applicants and other researchers for a process of isolating TCGF to substantial purity in a manner that is generally useful and also gives a high percentage recovery.
Several laboratories have reported partial purification of human TCGF from peripheral blood lymphocytes, Mier, J. W. et al., J. Immunol. 128, 1122-1127 (1982); Gillis, S., et al, Immunol. Rev. 63, 167-209 (1982); Robb, R. J. Immunobiol. 161, 21-50, (1982); from spleen leukocytes, Acuto, O., et al., J. Immunol. Methods 53, 15-26 (1982), and from the T-leukemia cell line, Jurkat, Gillis et al. supra; Robb, supra. However, in these literature reports, none of the compositions contains more than about 5 to 10 percent purity (as a weight percent of total protein).
Reindeau et al., The Journal of Biological Chemistry 258, 12114-12117 (1983) describe attempts to purify mouse interleukin-2. A C18 column was used in HPLC to purify IL-2 from EL4 cell secretions; however, as shown in FIG. 1, page 12115, there was no single elution peak, clearly evidencing that pure interleukin 2 was not obtained. Furthermore, recovery, even in this relatively impure condition, was only 70%. The paper also describes the synthesis of trace amounts of mouse IL-2 using mouse IL-2 messenger RNA to transcribe IL-2 in a cell-free wheat germ extract, followed by HPLC. Production of TCGF by messenger RNA transcription in this manner is not a practical method of producing useful amounts of TCGF.
Henricksen, O. and Frey, J., Cellular lmmunology 73 106-114 (1982) describe a fractionation of mouse IL-2 which is characterized as a "partial purification" in the abstract of the article. The stationary phase is not clearly specified in the article, as the stationary phase is referred to only by a tradename that is inclusive of several stationary phases produced by the same manufacturer. The graphic representation of the HPLC elution in FIG. 3, page 112, clearly indicates that the partial purification produces a far from homogeneous lL-2 preparation.
Henderson et al., The Journal of Immunology 131, 810-815, (1983) describe a partial purification of gibbon TCGF using HPLC. Although the process provides a relatively good recovery of TCGF activity, no homogeneous TCGF fraction is obtained, as clearly evidenced by the graphic representation of the HPLC elution in FIG. 4, page 812.
Milstone, D. S., and Parker, C. W., Biochemical and Biophysical Research Comm. 115, 762-768, (1983) describe a purification of gibbon ape TCGF, including a HPLC fractionation on a C18 column. Although TCGF fractions are characterized as being in "highly purified form", it is clear from the elution graphs that homogeneous TCGF is not obtained. From a reading of the article in its entirety, it can be estimated that the gibbon TCGF obtained is at best about 50% pure. Furthermore, the recovery of TCGF as noted on page 766 is only about 50%, confirming applicants' own experience with hydrophobic columns and the problem of excessive affinity of TCGF for highly hydrophobic columns. The paper lacks an amino acid analysis that would establish the degree of purity and lacks any other reliable data that establishes purity. SDS-PAGE data, as set forth in this paper, is a relatively unreliable indicator of purity because proteins of similar molecular wt. run together.
Recently, Robb, et al., Proc. Natl. Adac. Sci. USA 80 5990-5994 (1983) have obtained highly purified TCGF from Jurkat cells in an immunoaffinity chromatography procedure that utilizes monoclonal antibodies specific to TCGF; however, this technique requires large amounts of antibodies and may be difficult to adapt to large-scale TCGF purification. Previous data has raised speculation as to whether TCGF from tonsil cell-derived peripheral blood lymphocytes (tTCGF) differs from Jurkat cell-derived TCGF (jTCGF). Recently, the structure of a cDNA for human Jurkat cell-derived TCGF has been established, Taniguchi, T., Nature 302 305-310 (1983).
Because TCGF has important value for promoting cell growth in vitro and may be important as a therapeutic agent in vivo, it would be desirable to have a method of obtaining sizable amounts of substantially pure TCGF. This is particularly the case where the substance is to be administered to a human or other mammal as administration of unseparated impurities is to be avoided whenever possible.